Molecular analysis and mapping of gst genes
--L.Rossini, C.Frova, M.E.Pè and M.Sari Gorla
The glutathione(GSH)-glutathione-S-transferase(GST) system is a detoxification system capable of inactivating many toxic molecules. It is present in a wide variety of organisms, from mammals to insects and plants, in which it is involved in conferring tolerance to different classes of herbicides, including s-triazines, acetanilides and thiocarbamates. In maize, three gst genes (gst1, gstII, gstIII) (Shah et al., Plant Mol Biol 6:203-211, 1986; Moore et al. Nucleic Acids Res. 14:7277-7235,1986; A. Greenland, personal communication) have been identified and sequenced. [Ed. note: Temporary symbols pending clarifications of gene relationships.] They belong to a gene family and show extensive sequence homology at both DNA and protein levels. They encode for four isozymes which are active as dimers: GSTI, GSTIII and GSTIV are homodimers of 29, 26 and 27 kDa subunits respectively, while GSTII is a heterodimer of 29 (same as GSTI) and 27 (same as GSTIV) kDa subunits.
We found evidence that the number of maize gst genes and isozymes is higher, some isoforms being tissue-specific while others are expressed in most tissues. In particular, the two major bands detectable in electrophoretic enzyme assay (native PAGE) are expressed in roots, leaves and scutella. By screening numerous maize genotypes we have identified two inbred lines, B37 and B83, lacking these two bands in all tissues. Analysis of null/+ and null/null F1s indicated that the null mutations are recessive and allelic in the two inbreds. In order to identify to which of the characterized GST isoforms these bands correspond, we have carried out Northern experiments to analyze the transcription pattern of gstI and gstII in roots of normal and of the two null lines. As specific probes we have used a cDNA clone of gstII (kindly provided by A. Greenland) and a probe including the first exon of gstI synthesized via PCR on the basis of the published sequence information. gstII transcript was detected in all genotypes, while gstI mRNA was absent in the null lines. These data indicate that a single gene, gstI, controls the expression of the two isoforms. We tentatively identify them as GSTI (29 kDa homodimer) and GSTII (29/27 heterodimer).
The genomic organization of the two genes was investigated by Southern analysis: gst1 is a single copy gene, while the hybridization pattern of gstII suggests the presence of a duplicated gene. We have mapped these genes by RFLP analysis using an F2 population of 149 individuals, previously characterized for 110 molecular markers. gst1 was placed on the long arm of chromosome 8, while the two putative gstII loci, gstIIA and gstIIB, were mapped on chromosome 8 (70cM from gst1) and on chromosome 10 respectively (Figure).
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